PROJECT SUMMARY/ABSTRACT RNA functions as a central hub between DNA and protein, and understanding its regulation is critical to illuminating gene expression programs in normal and disease physiology. The properties of messenger RNA (mRNA) can be modulated by dynamic chemical modifications (the epitranscriptome) that occur post- transcriptionally, such as N6-methyladenosine (m6A), which regulates mRNA turnover, translation, nuclear export, and splicing, as well as other modifications on the nucleobases. A major challenge is to identify the functional consequences of these modifications and elucidate the molecular mechanisms by which they control mRNA biology. This proposal seeks to fill this knowledge gap by developing and applying chemical biology strategies to characterize proteins that mediate the effects of mRNA modifications on cellular processes. The epitranscriptome is shaped by RNA-modifying enzymes (writers and erasers) and interpreted by modification-specific RNA-binding proteins (readers). Characterizing these protein-RNA interactions is critical for understanding the function and regulation of specific modifications. Previously, we developed and applied a chemical proteomics strategy to profile binders of m6A. We identified new m6A readers as well as proteins that bind preferentially to unmodified RNA. Herein, we will interrogate the role of these m6A-mediated protein-RNA interactions on mRNA behavior in the cell and further develop our approach to profile readers of another methylation mark, N1-methyladenosine (m1A). Additionally, we propose novel methodologies to characterize modified RNA-protein interactions in vitro and in the cell. Our project has the following specific aims: Aim 1. Identify and functionally interrogate proteins that read mRNA methylation marks. We will focus on identifying and studying readers of m6A and m1A. Aim 2. Profile the substrate specificity of mRNA methylation readers and erasers by in vitro selection. Aim 3. Investigate the trafficking of methylated mRNA to cellular stress granules using an RNA proximity ligation strategy. Our findings will reveal how mRNA methylation regulates protein-RNA interactions to control gene expression. These studies should improve our understanding of fundamental RNA regulatory mechanisms and provide powerful and general strategies for interrogating the function of mRNA modifications.